Quadruplex DNA Structures Research Articles

A further contribution to the extreme variability of quadruplex structures from oligodeoxyribonucleotides containing inversion of polarity sites in the G-tract

Structural insight into DNA quadruplex structures formed by oligodeoxyribonucleotides 3'TG5'-5'GGGT3' (QS55) and 5'TG3'-3'GGGT5' (QS33) is presented. NMR analysis reveals that QS33 forms a parallel-like four-fold symmetric quadruplex, while QS55 possesses a two-fold symmetry and is characterized by a tetrameric antiparallel quadruplex embedded between two parallel tracts. The results reported here describe unprecedented quadruplex complexes provided by peculiar structural features never reported to date. These structures might inspire the design of new aptameric nucleic acids characterized by novel structural motifs hardly realizable with unmodified DNA/RNA.

Simulation of 125I-based radioprobing experiments to study DNA quadruplex structure and topology

Purpose: Certain guanine-rich DNA sequences have the capacity to fold into four-stranded structures stabilized by the stacking of square planar arrangements of four hydrogen-bonded guanine bases. However both the overall topology of folding and the more detailed three dimensional structure of these quadruplexes is difficult to determine or predict, and they can be polymorphic, altering radically depending on environmental conditions. Radioprobing experiments, in which Auger electrons emitted during the decay of a 125I-containing base induce strand cleavage in a distance- and structure-dependent manner, have provided possible means of determining these details. Here we have used a combination of computer simulation methods to study the information obtained by one such experiment, reported in 2004.Method: Models were constructed of three quadruplex topologies considered in the experiment, and one other topology proposed more recently. Molecular Dynamics simulations were used to equilibrate these structures and monitor how they evolved over several nanoseconds in solution. Snapshots from the trajectories were then subjected to Monte Carlo track structure prediction, from which theoretical cleavage patterns have been extracted.Results: The four topologies were found to yield quite different cleavage patterns, which allow the presence of particular conformations in an experiment to be predicted.Conclusion: Radioprobing, which is usable in biologically relevant environments, is sensitive enough to distinguish with some confidence between alternative folding topologies in a DNA structure. Monte Carlo track structure simulation can reinforce or question conclusions drawn from experiment, and Molecular Dynamics used with various restraints provides a practical means of guiding a model towards one that yields cleavage patterns closer to those found experimentally.

Structural and Thermodynamic Studies of the Interaction of Distamycin A with the Parallel Quadruplex Structure [d(TGGGGT)]4

The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by 1H NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)]4, we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGGMeGGT)]4, and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand-DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)]4 complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)]4 in a 4:1 binding mode, with two antiparallel distamycin dimers that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)]4 enhances ( approximately 10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groove-binder molecule complexed to a DNA quadruplex structure.

Structural insight into the h TERT intron 6 sequence d(GGGGTGAAAGGGG) from 1H-NMR study

The interest in DNA quadruplex structures has been fueled by the recognition that telomeres, the 3′ single stranded guanine-rich overhangs found at the termini of chromosomes, are likely to form G-tetrads type structures important in cell senescence and cancer. In addition to their presence in telomeres, where they may play a role in maintaining the stability and integrity of chromosomes, guanine-rich regions are found in other region of the genome, amongst these is intron 6 of hTERT a gene codifying for the enzyme telomerase. Interestingly, the formation of G-quadruplexes in this region is involved in the down-regulation of telomerase activity caused by an alteration of the hTERT splicing pattern. Therefore, we have analyzed several sequences of that intron by 1H-NMR and CD spectroscopy, and we have found that the sequence d(GGGGTGAAAGGGG) is able to fold in a single well-defined antiparallel quadruplex structure consisting of four G-tetrads, possessing a twofold symmetry, and containing four Gs in a syn glycosidic conformation.

A Triplex-forming Sequence from the Human c-MYC Promoter Interferes with DNA Transcription

Naturally occurring DNA sequences that are able to form unusual DNA structures have been shown to be mutagenic, and in some cases the mutagenesis induced by these sequences is enhanced by their transcription. It is possible that transcription-coupled DNA repair induced at sites of transcription arrest might be involved in this mutagenesis. Thus, it is of interest to determine whether there are correlations between the mutagenic effects of such noncanonical DNA structures and their ability to arrest transcription. We have studied T7 RNA polymerase transcription through the sequence from the nuclease-sensitive element of the human c-MYC promoter, which is mutagenic in mammalian cells (Wang, G., and Vasquez, K. M. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13448-13453). This element has two mirror-symmetric homopurine-homopyrimidine blocks that potentially can form either DNA triplex (H-DNA) or quadruplex structures. We detected truncated transcription products indicating partial transcription arrest within and closely downstream of the element. The arrest required negative supercoiling and was much more pronounced when the pyrimidine-rich strand of the element served as the template. The exact positions of arrest sites downstream from the element depended upon the downstream flanking sequences. We made various nucleotide substitutions in the wild-type sequence from the c-MYC nuclease-sensitive element that specifically destabilize either the triplex or the quadruplex structure. When these substitutions were ranked for their effects on transcription, the results implicated the triplex structure in the transcription arrest. We suggest that transcription-induced triplex formation enhances pre-existing weak transcription pause sites within the flanking sequences by creating steric obstacles for the transcription machinery.

Crystallographic studies of quadruplex nucleic acids

DNA quadruplexes are formed from guanine-rich repeats that self-associate into higher order four-stranded structures. These G-rich repeat sequences can be found in both telomeric regions as well as regions proximal to promoters of oncogenes. The compelling evidence that stabilizing these motifs by small molecule ligands can alter cell viability in certain cancer cell lines has led to identification of DNA quadruplex structures as therapeutic targets. Target-based design of selective ligands that target particular quadruplex topologies is heavily reliant on the availability of high-resolution structural information of the intended target. X-ray crystallography can provide this level of detail to atomic resolution. Recently drug discovery programs have refocused on the need for a fuller structural and molecular description of the target molecule. This review describes a crystallographic route to the determination of quadruplex topology, and high-resolution loop structures for target-based ligand design. The review also highlights the methods employed in the design of appropriate DNA sequences and crystallization techniques to solve these unusual DNA structures.

Synthesis and Characterization of Monomolecular DNA G-Quadruplexes Formed by Tetra-End-Linked Oligonucleotides

Guanine-rich DNA sequences are widely dispersed in the eukaryotic genome and are abundant in regions with relevant biological significance. They can form quadruplex structures stabilized by guanine quartets. These structures differ for number and strand polarity, loop composition, and conformation. We report here the syntheses and the structural studies of a set of interconnected d(TG(4)T) fragments which are tethered, with different orientations, to a tetra-end-linker in an attempt to force the formation of specific four-stranded DNA quadruplex structures. Two synthetic strategies have been used to obtain oligodeoxyribonucleotide (ODN) strands linked with their 3'- or 5'-ends to each of the four arms of the linker. The first approach allowed the synthesis of tetra-end-linked ODN (TEL-ODN) containing the four ODN strands with a parallel orientation, while the latter synthetic pathway led to the synthesis of TEL-ODNs each containing antiparallel ODN pairs. The influence of the linker at 3'- or 5'-ODN, on the quadruplex typology and stability, in the presence of sodium or potassium ions, has been investigated by circular dichroism (CD), CD thermal denaturation, (1)H NMR experiments at variable temperature, and molecular modeling. All synthesized TEL-ODNs formed parallel G-quadruplex structures. Particularly, the TEL-ODN containing all parallel ODN tracts formed very stable parallel G-quadruplex complexes, whereas the TEL-ODNs containing antiparallel ODN pairs led to relatively less stable parallel G-quadruplexes. The molecular modeling data suggested that the above antiparallel TEL-ODNs can adopt parallel G-quadruplex structures thanks to a considerable folding of the tetra-end-linker around the whole quadruplex scaffold.

Predictive modelling of topology and loop variations in dimeric DNA quadruplex structures

We have used a combination of simulated annealing (SA), molecular dynamics (MD) and locally enhanced sampling (LES) methods in order to predict the favourable topologies and loop conformations of dimeric DNA quadruplexes with T2 or T3 loops. This follows on from our previous MD simulation studies on the influence of loop lengths on the topology of intramolecular quadruplex structures [P. Hazel et al. (2004) J. Am. Chem. Soc., 126, 16 405–16 415], which provided results consistent with biophysical data. The recent crystal structures of d(G4T3G4)2 and d(G4BrUT2G4) (P. Hazel et al. (2006) J. Am. Chem. Soc., in press) and the NMR-determined topology of d(TG4T2G4T)2 [A.T. Phan et al. (2004) J. Mol. Biol., 338, 93–102] have been used in the present study for comparison with simulation results. These together with MM-PBSA free-energy calculations indicate that lateral T3 loops are favoured over diagonal loops, in accordance with the experimental structures; however, distinct loop conformations have been predicted to be favoured compared to those found experimentally. Several lateral and diagonal loop conformations have been found to be similar in energy. The simulations suggest an explanation for the distinct patterns of observed dimer topology for sequences with T3 and T2 loops, which depend on the loop lengths, rather than only on G-quartet stability.

Destabilization of Quadruplex DNA by 8‐Aminoguanine

Getting rid of unwanted quadruplexes. Oligonucleotides carrying 8-amino-2′-deoxyguanosine form very stable triple-helical structures. In this paper, we show that 8-amino-2′-deoxyguanosines destabilize quadruplex DNA structures. This quadruplex-destabilization effect is very convenient for the design of triplex-forming oligonucleotides, making 8-aminopurines superior to other purine derivatives. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2006/z500281_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

8-Methyl-2'-deoxyguanosine incorporation into parallel DNA quadruplex structures

This paper concerns the Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) structural studies of the quadruple helix arrangements adopted by three tailored oligodeoxyribonucleotide analogues, namely d(TGMeGGT), d(TGGMeGT) and d(TGGGMeT), where dGMe represents a 8-methyl-2′-deoxyguanosine residue. The results of this study clearly demonstrate that the effects of the incorporation of dGMe instead of a dG residue are strongly dependant upon the positioning of a single base replacement along the sequence. As such, d(TGMeGGT), d(TGGMeGT) have been found to form 4-fold symmetric quadruplexes with all strands parallel and equivalent to each other, each more stable than their natural counterpart. NMR experiments clearly indicate that [d(TGMeGGT)]4 possesses a GMe-tetrad with all dGMe residues in a syn-glycosidic conformation while an anti-arrangement is apparent for the four dGMe of [d(TGGMeGT)]4. As the two complexes show a quite different CD behaviour, a possible relationship between the presence of residues adopting syn-glycosidic conformations and CD profiles is briefly discussed. As far as d(TGGGMeT) is concerned, NMR data indicate that at 25°C it exists primarily as a single-strand conformation in equilibrium with minor amounts of a quadruplex structure.

Characterization of the Monovalent Ion Position and Hydrogen‐Bond Network in Guanine Quartets by DFT Calculations of NMR Parameters

Conformational stability of G-quartets found in telomeric DNA quadruplex structures requires the coordination of monovalent ions. Here, an extensive Hartree-Fock and density functional theory analysis of the energetically favored position of Li+, Na+, and K+ ions is presented. The calculations show that at quartet-quartet distances observed in DNA quadruplex structures (3.3 A), the Li+ and Na+ ions favor positions of 0.55 and 0.95 A outside the plane of the G-quartet, respectively. The larger K+ ion prefers a central position between successive G-quartets. The energy barrier separating the minima in the quartet-ion-quartet model are much smaller for the Li+ and Na+ ions compared with the K+ ion; this suggests that K+ ions will not move as freely through the central channel of the DNA quadruplex. Spin-spin coupling constants and isotropic chemical shifts in G-quartets extracted from crystal structures of K+- and Na+-coordinated DNA quadruplexes were calculated with B3LYP/6-311G(d). The results show that the sizes of the trans-hydrogen-bond couplings are influenced primarily by the hydrogen bond geometry and only slightly by the presence of the ion. The calculations show that the R(N2N7) distance of the N2-H2...N7 hydrogen bond is characterized by strong correlations to both the chemical shifts of the donor group atoms and the (h2)J(N2N7) couplings. In contrast, weaker correlations between the (h3)J(N1C6') couplings and single geometric factors related to the N1-H1...O6=C6 hydrogen bond are observed. As such, deriving geometric information on the hydrogen bond through the use of trans-hydrogen-bond couplings and chemical shifts is more complex for the N1-H1...O6=C6 hydrogen bond than for the N2-H2...N7 moiety. The computed trans-hydrogen-bond couplings are shown to correlate with the experimentally determined couplings. However, the experimental values do not show such strong geometric dependencies.

Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPalpha and TEBPbeta. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPalpha in attaching telomeres in the nucleus and in recruiting TEBPbeta to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPbeta in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle-dependent phosphorylation of TEBPbeta.

UNUSUAL MONOMOLECULAR DNA QUADRUPLEX STRUCTURES USING BUNCH-OLIGONUCLEOTIDES

The chemical synthesis of several G-rich bunch-oligonucleotides and the structural characterization of the corresponding monomolecular G-quadruplexes (I–IV) have been reported. The synthetic method allow the achievement of monomolecular DNA quadruplex structures having unusual and predeterminable oligodeoxyribonucleotide (ODN) strand orientation.

A new class of DNA quadruplexes formed by oligodeoxyribonucleotides containing a 3′-3′ or 5′-5′ inversion of polarity site

Unprecedented DNA quadruplex structures containing a 3'-3' or 5'-5' inversion of polarity site in the G-tract are presented; the quadruplexes are characterized by different elements of symmetry and glycosidic angle conformations.

A G-quadruplex telomere targeting agent produces p16-associated senescence and chromosomal fusions in human prostate cancer cells

The trisubstituted acridine derivative BRACO-19 has been designed to interact with and stabilize the quadruplex DNA structures that can be formed by folding of the single-stranded repeats at the 3' end of human telomeres. We suggest that the BRACO-19 complex inhibits the catalytic function of telomerase in human cancer cells and also destabilizes the telomerase-telomere capping complex so that cells enter senescence. Here, we present evidence showing that the inhibition of cell growth caused by BRACO-19 in DU145 prostate cancer cells occurs more rapidly than would be expected solely by the inhibition of the catalytic function of telomerase, and that senescence is accompanied by an initial up-regulation of the cyclin-dependent kinase inhibitor p21, with subsequent increases in p16(INK4a) expression. We also show that treatment with BRACO-19 causes extensive end-to-end chromosomal fusions, consistent with telomere uncapping.

Detection of quadruplex DNA structures in human telomeres by a fluorescent carbazole derivative.

Single-stranded telomeric DNA tends to form a four-base-paired planar structure termed G-quadruplex. This structure was easily formed in vitro in the presence of monovalent cations. However, the existence of this structure in native human telomeres is unclear. Here we address this important question through the distinctive properties of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) upon binding to various DNA structures. Although the fluorescence of BMVC increases significantly in the presence of DNA, BMVC has high sensitivity and binding preference to quadruplex d(T(2)AG(3))(4) over duplex DNA. In addition, the fluorescent emissions were characterized around 575 nm for quadruplex d(T(2)AG(3))(4) and 545 nm for most of duplex DNA. The 575-nm fluorescence emissions were detected in the mixtures of 2 nM BMVC with the chromosomal DNA that were extracted from human cells, suggesting the presence of quadruplex structure in human nucleus. Further analyzing the BMVC fluorescence at the ends of metaphase chromosomes and other regions of chromosomes, we detected the quadruplex-binding BMVC fluorescence at telomere-proximal regions. Together these results provide the first evidence for the presence of quadruplex structures in human telomeres.

NMR solution structure of a parallel LNA quadruplex.

The solution structure of a locked nucleic acid (LNA) quadruplex, formed by the oligomer d(TGGGT), containing only conformationally restricted LNA residues is reported. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the complex. The molecule adopts a parallel stranded conformation with a 4-fold rotational symmetry, showing a right-handed helicity and the guanine residues in an almost planar conformation with three well-defined G-tetrads. The thermal stability of Q-LNA has been found to be comparable with that of [r(UGGGU)]4, while a T(m) increment of 20 degrees C with respect to the corresponding DNA quadruplex structure [d(TGGGT)]4 has been observed. The structural features of the LNA quadruplex reported here may open new perspectives for the biological application of LNAs as novel versatile tools to design aptamer or catalyst oligonucleotides.

NMR Structure of the Thrombin-Binding DNA Aptamer Stabilized by Sr2+

The structure of thrombin-binding DNA aptamer complexed with a single Sr2+ ion (Sr2+:TBA complex) has been determined using NMR spectroscopy and restrained molecular dynamics simulations. The quadruplex structure for the Sr2+:TBA complex is similar in topology, but distinct in structure, from that previously reported for the K+:TBA complex. The inter-tetrad distance of the Sr2+:TBA complex is 3.8 Å, or 0.7 Å larger than in the K+:TBA complex. This substantial difference can be attributed to a different binding site for Sr2+ in the Sr2+:TBA complex than for K+ in the K+:TBA complex. The Sr2+:TBA complex assumes a 1:1 stoichiometry, and it is very likely that the Sr2+ ion simultaneously interacts with the eight O6 atoms of the two G-tetrads. The results indicate that quadruplex DNA structures are highly sensitive to the presence of specific metal ions. The binding of specific metal ions may modulate the biological activity of quadruplex DNA structures in vivo.

Trisubstituted acridine derivatives as potent and selective telomerase inhibitors.

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.

The stability of intramolecular DNA quadruplexes with extended loops forming inter- and intra-loop duplexes.

We have examined the formation of intramolecular quadruplex DNA structures in which the loops have been extended so as to generate short DNA duplexes. Fluorescence melting and DNase I cleavage studies show that duplexes can be formed within each loop, but that duplexes between the loops are not stable.